Abstract Infection with the protozoan parasite Trypanosoma cruzi is generally controlled but often not eliminated by host immune responses. In humans and many other hosts, this persistent infection ultimately results in muscle tissue damage known as Chagas disease. Although several partially effective drugs exist to treat the infection, it is estimated that only ~1% of infected subjects receive treatment. The main barrier to the wider use of current drugs and the development of better therapeutics in Chagas disease is the absence of reliable methods to definitively determine the efficacy of treatment. The goal of this project is to validate and improve tests of cure for T. cruzi infection, relying primarily on the changing pattern of antibody responses to a panel of defined T. cruzi antigens following effective treatment. Our previous ~10 year have validated this approach using a limited set of antigens using a robust, but expensive Luminex bead-based approach. In this project, we will use protein and glycan microarrays to select additional high quality targets for measurement of anti-T. cruzi antibodies, expanding from the current 15 parameters in the Luminex assay to 50 or more parameters. This increase in target number and diversity will improve the quality and the speed of determination of treatment efficacy and assure that all exposed subjects are identified. Using this expanded panel of T. cruzi antigens and the much reduced cost of testing provided by an array format, we will develop and validate a test of cure. The initial test parameters (e.g. the number and selection of target antigens, proteins and/or glycans, and number of serial samples required and over what follow-up period) will be established using sera from long-term infected macaques treated with benznidazole and sampled multiple times before and at 6 week intervals after treatment for up to a year post-treatment, at which time the cure status is definitively determined as assisted by immunosuppression. These parameters will be validated using sera serially sampled from humans for up to 15 years post-treatment wherein the conversion to negative conventional serology, the currently accepted measure of cure, will be applied. Finally, we will apply the validated test prospectively to subjects newly enrolled in treatment programs in 2 sites in Argentina and 1 in Brazil. The overall goal of this process will be to identify the fewest number of samples collected per patient over the shortest period of time post- treatment that are needed to discriminate between treatment success and failure using the fewest number of target antigens. Using a combination of historical stored samples and recent samples from the same subjects and the test of cure, we will also assess the rate of spontaneous cure in untreated subjects. Identification of seropositive persons who have spontaneously resolved the infection is important for prognosis /counseling and to prevent their exposure to treatment and its common accompanying adverse complications. Completion of these studies will allow improved selection of candidates for treatment, assessment of outcomes in those subjects who receive treatment, and provide the critical tool needed to identify candidate drugs that perform better and are safer than the currently available, but underutilized compounds.